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Down-regulation of nicotinamide N-methyltransferase induces apoptosis in human breast cancer cells via the mitochondria-mediated pathway.

Zhang J, Wang Y, Li G, Yu H, Xie X - PLoS ONE (2014)

Bottom Line: Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies.The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT.In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Sir RunRun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

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Construction of MCF-7 and SK-BR-3 cell strains expressing NNMT stably.Real-Time RT-PCR analysis (A, C) and Western blot (B, D) were used to analyze NNMT expression in MCF-7, MCF-7/Vector, MCF-7/NNMT-1, MCF-7/NNMT-2, SK-BR-3, SK-BR-3/Vector, SK-BR-3/NNMT-1 and SK-BR-3/NNMT-2. GAPDH was used as an internal control. (A, B) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in MCF-7 cells compared to the MCF-7/Vector. (C, D) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in SK-BR-3 cells compared to the SK-BR-3/Vector. The differences between cells transfected with pcDNA3.1 and wild type cells were not significant both in MCF-7 cells and SK-BR-3 cells. (B) and (D) shows the protein quantification of the western blot results, respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (B, D) are expressed as means ± SD of four independent experiments. *P<0.05 vs. control group.
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pone-0089202-g007: Construction of MCF-7 and SK-BR-3 cell strains expressing NNMT stably.Real-Time RT-PCR analysis (A, C) and Western blot (B, D) were used to analyze NNMT expression in MCF-7, MCF-7/Vector, MCF-7/NNMT-1, MCF-7/NNMT-2, SK-BR-3, SK-BR-3/Vector, SK-BR-3/NNMT-1 and SK-BR-3/NNMT-2. GAPDH was used as an internal control. (A, B) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in MCF-7 cells compared to the MCF-7/Vector. (C, D) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in SK-BR-3 cells compared to the SK-BR-3/Vector. The differences between cells transfected with pcDNA3.1 and wild type cells were not significant both in MCF-7 cells and SK-BR-3 cells. (B) and (D) shows the protein quantification of the western blot results, respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (B, D) are expressed as means ± SD of four independent experiments. *P<0.05 vs. control group.

Mentions: The Bcl-2 family of proteins, which shares homology in any of the four common Bcl-2 homology (BH) domains, was highly related with apoptosis [29]. Thus, we analyzed the changes of Bcl-2 family and found that the expression of Bax, Bcl-2, Bcl-xL and Puma significantly changed in NNMT down-regulated cells. As shown in Figure 7, the expression of pro-apoptotic proteins, Bax and Puma, was up-regulated significantly in both cell lines infected with NNMT shRNA 1# and NNMT shRNA 2# (p<0.01). On the contrary, the expression of Bcl-2 and Bcl-xL, which are identified as anti-apoptotic proteins, was significantly down-regulated (p<0.01). As a result, the down-regulation of NNMT increased both mRNA and protein ratio of Bax/Bcl-2 compared to negative control (p<0.01). This result indicated that apoptosis might be induced by down-regulation of NNMT via regulating the Bax/Bcl-2 ratio in human breast cancer cells.


Down-regulation of nicotinamide N-methyltransferase induces apoptosis in human breast cancer cells via the mitochondria-mediated pathway.

Zhang J, Wang Y, Li G, Yu H, Xie X - PLoS ONE (2014)

Construction of MCF-7 and SK-BR-3 cell strains expressing NNMT stably.Real-Time RT-PCR analysis (A, C) and Western blot (B, D) were used to analyze NNMT expression in MCF-7, MCF-7/Vector, MCF-7/NNMT-1, MCF-7/NNMT-2, SK-BR-3, SK-BR-3/Vector, SK-BR-3/NNMT-1 and SK-BR-3/NNMT-2. GAPDH was used as an internal control. (A, B) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in MCF-7 cells compared to the MCF-7/Vector. (C, D) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in SK-BR-3 cells compared to the SK-BR-3/Vector. The differences between cells transfected with pcDNA3.1 and wild type cells were not significant both in MCF-7 cells and SK-BR-3 cells. (B) and (D) shows the protein quantification of the western blot results, respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (B, D) are expressed as means ± SD of four independent experiments. *P<0.05 vs. control group.
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pone-0089202-g007: Construction of MCF-7 and SK-BR-3 cell strains expressing NNMT stably.Real-Time RT-PCR analysis (A, C) and Western blot (B, D) were used to analyze NNMT expression in MCF-7, MCF-7/Vector, MCF-7/NNMT-1, MCF-7/NNMT-2, SK-BR-3, SK-BR-3/Vector, SK-BR-3/NNMT-1 and SK-BR-3/NNMT-2. GAPDH was used as an internal control. (A, B) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in MCF-7 cells compared to the MCF-7/Vector. (C, D) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in SK-BR-3 cells compared to the SK-BR-3/Vector. The differences between cells transfected with pcDNA3.1 and wild type cells were not significant both in MCF-7 cells and SK-BR-3 cells. (B) and (D) shows the protein quantification of the western blot results, respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (B, D) are expressed as means ± SD of four independent experiments. *P<0.05 vs. control group.
Mentions: The Bcl-2 family of proteins, which shares homology in any of the four common Bcl-2 homology (BH) domains, was highly related with apoptosis [29]. Thus, we analyzed the changes of Bcl-2 family and found that the expression of Bax, Bcl-2, Bcl-xL and Puma significantly changed in NNMT down-regulated cells. As shown in Figure 7, the expression of pro-apoptotic proteins, Bax and Puma, was up-regulated significantly in both cell lines infected with NNMT shRNA 1# and NNMT shRNA 2# (p<0.01). On the contrary, the expression of Bcl-2 and Bcl-xL, which are identified as anti-apoptotic proteins, was significantly down-regulated (p<0.01). As a result, the down-regulation of NNMT increased both mRNA and protein ratio of Bax/Bcl-2 compared to negative control (p<0.01). This result indicated that apoptosis might be induced by down-regulation of NNMT via regulating the Bax/Bcl-2 ratio in human breast cancer cells.

Bottom Line: Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies.The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT.In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Sir RunRun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

Show MeSH
Related in: MedlinePlus